The application concerns an analytical element for determining the amount of an analyte in a liquid containing a sample application zone and a detection zone which are in liquid-transferring contact, the latter containing at least one enzyme and an indicator substance, the enzyme catalysing a reaction in which the analyte or a substance derived from the analyte participates and the indicator substance forming a signal when the analyte is present which signal correlates with the amount of analyte, and containing a liquid-permeable interference-reducing layer in direct contact with the detection zone arranged such that liquid does not reach the detection zone until it has passed through the interference-reducing layer, the interference-reducing layer containing at least one enzyme which participates in a reaction of the analyte to be determined or of a substance derived from the analyte. The application additionally concerns a method for the determination of an analyte in a liquid by means of such a multilayer analytical element.
Results for analytes to be detected that are too high i.e. false positive often occur with analytical elements of the dry chemistry type which are often called test strips on which undosed sample volumes and hence volumes which vary widely are applied. In addition it is often not possible to recognize when the detection reaction is completed. The detection reaction often slowly declines over a long time period (reaction creep).
The Japanese laid-open specification No. Hei 5-23199 (publication date Feb. 2, 1993) describes an analytical element and a corresponding analytical method for analysing neutral fats. The object of the invention described in the patent application is to remove glycerol that was originally present in the samples to be examined before carrying out an enzymatic analysis of neutral fats via glycerol as an intermediate step. This is achieved by providing analytical elements for the analysis of neutral fats which have at least one reagent layer on a transparent support and a reaction layer thereon. The reaction layer which is the first layer to come into contact with the sample to be examined contains glycerol dehydrogenase and NAD+. The reagent layer also contains glycerol dehydrogenase but no co-enzyme. Consequently glycerol is converted into dihydroxyacetone and NADH in the reaction layer which acts as an interference-reducing layer and is thereby removed. Apparently the NAD+ which diffuses together with the sample from the reaction layer into the reagent layer, activates the glycerol dehydrogenase that is present there such that glycerol formed in the course of the analytical reaction of the neutral fats is also converted in the reagent layer into dihydroxy-acetone and NADH. The NADH that is formed reacts with a chromogen in the reagent layer to form a coloured detection product. It is to be expected that NADH which diffuses from the reaction layer into the reagent layer during the course of the analysis would influence the colour reaction in a false-positive manner.
In view of this prior art it was regarded as an object to provide analytical elements which also give correct results with undosed samples i.e. results which agree with those obtained with the respective reference methods. In addition the result should be available after a short time. It should be possible to clearly recognize the end of the detection reaction by the fact that there is no further substantial change in a signal that is used to determine an analyte.
This object is achieved according to the invention by the subject matter which is characterized in more detail in the patent claims.
The invention concerns in particular an analytical element for determining the amount of an analyte in a liquid containing a sample application zone and a detection zone which are in liquid-transferring contact, the latter containing at least one enzyme and an indicator substance, the enzyme catalysing a reaction in which the analyte or a substance derived from the analyte participates and the indicator substance forming a signal when the analyte is present which signal correlates with the amount of analyte, and containing a liquid-permeable interference-reducing layer in direct contact with the detection zone arranged such that liquid does not reach the detection zone until it has passed through the interference-reducing layer, the interference-reducing layer containing at least one enzyme which participates in a reaction of the analyte to be determined or of a substance derived from the analyte, characterised in that the analyte or the substance derived from the analyte is converted enzymatically in the interference-reducing layer into end products which cannot contribute to the signal formation by the indicator substance.
The invention also concerns a method for the determination of an analyte in a liquid by means of an analytical element described above characterised in that liquid is applied to the sample application zone in an undosed manner, liquid passes through the interference-reducing layer into the detection zone and there the amount of analyte in the liquid is determined in this process the detection zone is filled with liquid and excess liquid remains in the interference-reducing layer and optionally in the sample application zone.
A subject matter of the invention is especially the use of a layer in a multilayer analytical element which converts an analyte to be determined or a substance derived therefrom into products which do not contribute to the signal formation in this multilayer analytical element for the determination of this analyte, to prevent rediffusion of analyte from other zones into the zone of the analytical element in which it is intended to detect the analyte when the detection zone is filled with liquid.
An analytical element according to the invention contains a matrix material which contains a sample application zone and a detection zone. Several matrix materials may also be present one of which carries a sample application zone and the other carries the detection zone. All absorbent or swellable materials which can imbibe a liquid can basically be used as matrix materials. These can be fibrous materials such as fleeces, fabrics or knitted fabrics or non-fibrous materials such as porous or non-porous films or membranes.
In an analytical element according to the invention the sample application zone and the detection zone are in a liquid-transfer-enabling contact. The sample application zone can touch the detection zone. However, both zones can also be separate provided that liquid which is applied to the sample application zone can pass into the detection zone in the analytical element.
In order to improve the handling of the analytical element, the sample application zone and the detection zone can be disposed on an inert stiff support material. All inert, adequately stiff materials such as glass, hydrophobised cardboard or polymer materials are potentially suitable for such a support material. Stiff polymer foils which are for example composed of methacrylate/acrylate, polystyrene or polycarbonate are preferably used. In addition the support material can be transparent or impermeable to light.
In an analytical element according to the invention the sample application zone and detection zone can be arranged next to one another or above one another.
The sample application zone refers to the area of the analytical element according to the invention on which the liquid sample is applied. The detection zone of the analytical element according to the invention is understood as the area in which, in the presence of the analyte, a signal is produced which correlates with the amount of the analyte.
The reagents required to determine the amount of an analyte can be distributed over several layers in the detection zone. This has proven to be especially advantageous when certain reagents are not compatible with one another and thus cannot be accommodated within one layer. The detection zone contains at least one enzyme and an indicator substance which, as described above, can both be present within or on a layer or can be located in or on several layers. In this connection an enzyme is understood as a protein which, together with a prosthetic group or a co-enzyme, catalyses a reaction in which the analyte or a substance derived from the analyte participates. In a glucose test the enzyme can for example be glucose oxidase. When detecting triglycerides this enzyme can for example also be glycerokinase which catalyses a reaction of the substance glycerol derived from the analyte triglyceride. Substances derived from the analyte are usually those that have been formed from the substance to be determined by means of an enzymatic or non-enzymatic reaction and whose amount can be correlated with the amount of the analyte.
Indicator substances refer to those materials which produce a signal with a substance derived from the analyte which correlates with the amount of analyte. Suitable indicator substances according to the invention are in particular those which produce a colour, lose their colour or change their colour or which lose fluorescence or generate fluorescence in the presence of the analyte when the analytical element according to the invention is used. However, those indicator substances are preferred according to the invention which produce a colour, lose their colour or change their colour. Such substances are also referred to as chromogens.
The analytical element according to the invention contains a liquid-permeable interference-reducing layer in direct contact with the detection zone. The interference-reducing layer is arranged such that liquid only reaches the detection zone after passing through the interference-reducing layer. In this connection the interference-reducing layer and detection zone can be arranged next to one another or above one another. The interference-reducing layer can represent the sample application zone when liquid sample is applied directly to the interference-reducing layer. The interference-reducing layer and sample application zone can, however, also be spatially separate from one another. Thus the sample application zone and interference-reducing layer can also be stacked on top of one another or be arranged next to one another if the sample application zone and interference-reducing layer are not identical.
According to the invention the interference-reducing layer contains at least one enzyme which catalyses a. reaction of the analyte to be determined or of a substance derived from the analyte to obtain end products which cannot contribute to the signal generation by the indicator substance. Consequently end products are obtained according to the invention in the interference-reducing layer from the analyte or from a substance derived from the analyte at the end of the enzymatic interference-reducing reaction which cannot generate a signal with the indicator substance. If for example the indicator substance together with hydrogen peroxide forms a dye in the presence of peroxidase, then the analyte or the substance derived from the analyte must be enzymatically converted in the interference-reducing layer in such a manner that ultimately no hydrogen peroxide remains in the interference-reducing layer. Should hydrogen peroxide be formed enzymatically in the interference-reducing layer, this must be enzymatically reacted further in the interference-reducing layer such that hydrogen peroxide does not remain in the interference-reducing layer as an end product. For example a reaction producing hydrogen peroxide can be coupled with a reaction consuming hydrogen peroxide. Catalase or peroxidase can be used for this as proteins that decompose hydrogen peroxide.
If an indicator substance is used in the detection zone which is accepted instead of oxygen as the electron-accepting substance by redox enzymes such as oxidases, it is possible to place a hydrogen peroxide producing reaction system in the interference-reducing layer. Substances which are accepted by redox enzymes as electron-accepting substances instead of oxygen are for example described in the European Patent Application 0 354 441. Such substances are not influenced further by hydrogen peroxide.
If a chromogen is used as the indicator substance, it is obvious that those end products are formed in the interference-reducing layer which do not interfere with the colour formation or colour change of the indicator substance. If the indicator substance forms a colour in the presence of the analyte, it is advantageous when the end products formed in the interference-reducing layer are colourless.
If a co-enzyme-dependent enzyme is used in the enzyme-containing layer of the detection zone, the required co-enzyme is preferably located in the analytical element according to the invention in the same layer of the detection zone.
Whereas the reagents required to determine the analyte can be separated into several layers of the detection zone as described above, it has proven to be particularly advantageous when the interference-reducing layer and a layer of the detection zone containing enzyme and indicator substance are arranged to be in direct contact with one another in the analytical element according to the invention. Under certain circumstances the substances required to determine the analyte that are not accommodated in the same layer of the detection zone containing the enzyme and/or indicator substance can also be located in the interference-reducing layer.
As already mentioned above the interference-reducing layer can also be the sample application zone if liquid sample is applied directly to the interference-reducing layer. However, it may also be expedient to not directly apply the sample to be determined to the interference-reducing layer but rather to a sample application zone that is located in front of the interference-reducing layer. This may then be particularly advantageous when for example it is intended to examine liquids containing particles such as blood. In such cases it has proven to be preferable to separate and retain the particles that are present in the liquid, such as blood cells like erythrocytes, in the sample application zone before the remaining liquid passes into the interference-reducing layer. In such cases it has proven to be advantageous to use fibrous matrix materials and especially fleeces and very preferably glass fibre fleeces as described for example in the European Patent Application 0 045 476.
In a determination method according to the invention an analytical element as described above is used. Liquid is preferably applied unmetered to the sample application zone. This is possible when a matrix material is used as the detection zone which can imbibe an exactly defined liquid volume. Such materials are known to a person skilled in the art. For example membranes are commercially available which fulfil these conditions. Films can, however, also be used as known for example from the European Patent Application 0 016 387.
Liquid passes from the sample application zone through the interference-reducing layer into the detection zone where the indicator substance produces a signal when the analyte is present which correlates with the amount of analyte. The signal can be measured by an apparatus. In the case of colour formation, colour reduction or colour change the signal can be measured by an instrument e.g. by reflection photometry or it can be determined visually.
The method according to the invention for the determination of an analyte in a liquid by means of an analytical element according to the invention can be carried out advantageously especially when so much liquid has been applied to the sample application zone of the analytical element that excess liquid remains in the interference-reducing layer and optionally in the sample application zone. In such cases false-positive results are often obtained with analytical elements of the prior art or the end of the signal generating reaction is not clearly discernible because analyte rediffuses from the excess liquid into the detection zone. In contrast the method according to the invention and the analytical element according to the invention can be particularly advantageously employed when using undosed liquid samples since falsely elevated results are not found and the end of the detection reaction can be clearly recognized since after a certain time point there is no further significant change in signal. Results are obtained with the method according to the invention and analytical element according to the invention which correlate well with those of standard wet chemistry methods. This applies in particular to the determination of trigylcerides and glucose.
The advantages of the analytical element according to the invention and of the method according to the invention are primarily achieved by using a layer in a multilayer analytical element which converts an analyte to be determined or a substance derived therefrom into products which do not contribute to the signal generation in the multilayer analytical element. This prevents rediffusion of analyte from other zones into the zone of the analytical element in which the detection of the analyte is to take place. This effect occurs in particular when the detection zone is filled with liquid and excess liquid is still present in other zones of the analytical element.